The effects of mutation on the neuroinvasiveness of Sindbis virus (SB) in a suckling mouse model system will be investigated. Attenuated mutants (prototype SB-RL) have been isolated by serial passage under selection for rapid growth in cell culture. SB-RL did not invade the brain as rapidly as the virulent wild-type, nor did it replicate as extensively in brain tissue. To explore the molecular basis for this difference in neuroinvasiveness, a number of virulent and attenuated strains have been compared, and virulent revertants of SB-RL have been characterized. An alteration in virion topography, identified with neutralizing monoclonal antibodies, was linked to reduced neuroinvasiveness in animals as well as to rapid penetration of cultured cells. The specific aims of the proposed research are 1) to determine the primary sites of mutations which lead to attenuation and 2) to define the virion rearrangements which result from such mutations. The first goal will be accomplished by cloning and sequencing the structural protein coding regions of the prototype virulent and attenuated genomes. Putative sites for the causal mutation will be confirmed by sequencing selected genome regions of other virulent and attenuated strains as well as spontaneous and monoclonal antibody selected virulent revertants. A similar approach will be employed at the protein level using tryptic peptide analysis and protein sequencing. The second goal will be attained by probing the surfaces of virulent, attenuated and revertant virions with monoclonal antibodies, proteases, and specific amino acid reactive reagents. Because closely related mutants and their revertants will be used to define mutations that affect penetration in cell culture as well as neurovirulence in vivo, it should be possible to assign a functional role to specific glycoprotein domains in both of these important and complex biological processes.